Tumor cells make mutations, creating genetic variants that adapt to stressful environments. The majority of cancer driver mutations may be caused by replication errors, but the sources and mechanisms of such replication errors in human tumors are poorly understood. This critical gap in knowledge hinders our ability to effectively manage cancer. The human genome is characterized by DNA sequence complexity and high repetitive DNA content, but we lack detailed mechanisms as to how this complex genome is replicated, limiting our understanding of the endogenous processes that promote cancer development. DNA polymerases encounter many Difficult-To-Replicate Sequences, or DiToRS, within repetitive regions that cause replication fork stalling. If not navigated efficiently, DiToRS lead to double strand breaks and genome instability. Our long-term goals are to illuminate mechanisms of human genome replication and expand knowledge of replication errors in tumor evolution. This project will discover how human cells carry out essential synthesis of DiToRS genome-wide, and the genetic consequences of incomplete DiToRS replication. Our established interdisciplinary team has unique expertise in studying DiToRS regions of the human genome, and has made important mechanistic advances towards understanding DiToRS replication. Our recent studies implicate DNA polymerases eta (Pol ?) and kappa (?) as being essential for this process, and advance a new paradigm in which Pols ? and ? have adopted critical functions for complex genome replication. Because these polymerases have low fidelity, we hypothesize that by requiring cells to engage Pols ?/? in DiToRS replication, replication stress increases replication errors in tumor cells. To test this innovative hypothesis, we will use human cell models and a physiologic source of replication stress linked to oncogene activation: dNTP substrate depletion. Aim 1 will study DNA polymerase biochemistry and identify key replication proteins required for DiToRS replication. Aim 2 will investigate the critical Pol ? functions required to promote tumor cell survival in the presence of replication stress, and the impact of Pol ? on ATR/Chk1 inhibitor targeted therapies. Aim 3 will measure mutations arising in non-tumorigenic and tumorigenic cells under stress, and develop new computational tools to interrogate cancer genomes for replication errors. Our mechanistic studies will provide key insights into the mechanisms by which oncogene activation results in replication errors that drive tumorigenesis, and reveal new biomarkers of tumor progression. Insights into mutational mechanisms gained from this project can be leveraged in precision oncology approaches to cancer therapy.